The upregulation of gill Na+/K+-ATPase activity is considered critical for the successful acclimation of salmonid fishes to seawater. The present study examines the mRNA expression of two recently discovered alpha-subunit isoforms of Na+/K+-ATPase (alpha1a and alpha1b) in gill during the seawater acclimation of three species of anadromous salmonids, which vary in their salinity tolerance. Levels of these Na+/K+-ATPase isoforms were compared with Na+/K+-ATPase activity and protein abundance and related to the seawater tolerance of each species. Atlantic salmon (Salmo salar) quickly regulated plasma Na+, Cl- and osmolality levels within 10 days of seawater exposure, whereas rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) struggled to ionoregulate, and experienced greater perturbations in plasma ion levels for a longer period of time. In all three species, mRNA levels for the alpha1a isoform quickly decreased following seawater exposure whereas alpha1b levels increased significantly. All three species displayed similar increases in gill Na+/K+-ATPase activity during seawater acclimation, with levels rising after 10 and 30 days. Freshwater Atlantic salmon gill Na+/K+-ATPase activity and protein content was threefold higher than those of Arctic char and rainbow trout, which may explain their superior seawater tolerance. The role of the alpha1b isoform may be of particular importance during seawater acclimation of salmonid fishes. The reciprocal expression of Na+/K+-ATPase isoforms alpha1a and alpha1b during seawater acclimation suggests they may have different roles in the gills of freshwater and marine fishes; ion uptake in freshwater fish and ion secretion in marine fishes.